ras protooncogenes are activated by characteristic point mutations in a wide variety of malignancies. The expressed p21 ras proteins are oncogenic by virtue of single substituted amino acids, usually at position 12 or 61 of the 189-residue p21 ras gene product. ras proteins act as membrane-associated molecular switches that bind GTP and GDP and slowly hydrolyze GTP to GDP.
Mutations in ras are associated with the vast majority of adenocarcinomas of the colon. Cancer of the colon is a highly treatable and often curable disease when it remains localized to the bowel. It is the second most frequently diagnosed malignancy in the United States as well as the second most common cause of cancer death. Surgery is the primary treatment and results in cure in approximately 50% of patients. Adenocarcinoma is the primary lesion in the majority of cases. Recurrence following surgery is a major problem and often is the ultimate cause of death. The prognosis for colon cancer patients is clearly related to the degree of penetration of the tumor through the bowel wall and the presence or absence of nodal involvement. For locally advanced disease, the role of radiation therapy in colon cancer is under clinical evaluation. There is no standard therapy for advanced colon cancer and no evidence that chemotherapy improves survival, although short-term palliation may be achieved in approximately 10-20% of patients.
Pancreatic carcinoma has a high incidence of K-ras mutations. Mutated K-ras sequences which can be identified by polymerase chain reaction utilizing allele-specific primers can even be found in the plasma or serum from patients with pancreatic carcinoma. The c-Ki-ras oncogene is activated by point mutations involving codon 12 in 72%-100% of primary pancreatic adenocarcinomas, but the gene is not activated in nonneoplastic tissues. Cancer of the exocrine pancreas is rarely curable. The highest cure rate (4%-12%) occurs if the tumor is truly localized to the pancreas. Unfortunately, this stage of disease accounts for fewer than 20% of cases and, even with surgical resection, results in little more than a 5% 5-year survival rate. For small cancers (less than 2 cm) in the head of the pancreas with no lymph node metastases and no extension beyond the "capsule" of the pancreas, the survival rate following resection of the head of the pancreas approaches 20%. Overall survival rate of all stages is less than 2% at 5 years with most patients dying within one year. Worldwide, very few patients with cancers of the pancreatic tail or uncinate process have been cured.
Lung cancers also frequently involve ras mutations. Point mutations in codon 12 of the K-ras protooncogene occur more frequently in lung adenocarcinomas from smokers (30%) than they do in lung adenocarcinomas from nonsmokers (7%), suggesting that smoking is an important factor in the induction of these mutations. The ras oncogene may thus be a specific target of the mutagenic activity of tobacco smoke, and suggest that DNA alterations at this site can occur early and irreversibly during the development of adenocarcinomas of the lung.
Mutations in the ras protooncogenes are the most frequently observed molecular alteration in acute myeloid leukemia (AML). Whether ras mutations occur as late or relatively early events in the multistep process of myeloid transformation, remains an open question. There is significant evidence that the ras oncogene plays a role in experimental mammary carcinogenesis; the evidence in human breast cancer, however, is more limited.
Similarly, there is significant evidence that the ras oncogene plays a role in nitrosoamine-induced esophageal tumors in rats, but in human esophageal cancers ras gene mutations are more rarely found. However, it is probable that there is a significant role of mutated ras genes in both cell proliferation and malignant transformation of human esophageal cells.
Certain human neuroblastomas also show a high incidence of oncogenic ras mutations. Indeed, one study suggested that expressions of the oncogene N-myc and p21 together as detected by immunohistochemical staining could be among the most reliable prognostic indicators in neuroblastoma patients.
The ras proteins are key regulators of the growth of eukaryotic cells. Some of the direct targets are unknown. These target proteins include raf-1, gap, phosphatidylinositol-3-hydroxykinase and, very recently, two nuclear proteins, C-JUN and its kinase (JNK) The three-dimensional x-ray crystal structure for a ras-related protein bound to a domain of raf-1 has been elucidated. The ras-related protein (rak-1-a) binds to raf directly, utilizing residues contained in a sequence involving amino acids 35-37. All of the contact residues in the ras-related protein are homologous to those in the corresponding segment of ras-p-21. One of the inventors has shown that the p-21 ras protein (35-47 segment) selectively inhibits the mitogenic effects of oncogenic ras-p-21.
In addition to its role as an oncogene, the activation of ras proteins is a key step in the signal transduction pathways triggered by ligand-bound cell surface receptors, such as the insulin receptor.
The classical target of the ras protein is the GTPase activating protein GAP. This target protein is thought to play an essential role in the regulation of ras activity by increasing the GTPase activity of wild type, but not transformed ras. On the other hand, there is a considerable superfamily of these GAP-related proteins, which includes p120-GAP. Other target proteins besides mammalian gap itself include (1) IRA1 and IRA2, the functional equivalents of GAP in yeast. They regulate the ras-cyclic AMP pathway, controlling cell growth; (2) sar1, the fission yeast protein that regulates ras1 in that organism; (3) BUD2, a yeast protein that activates BUD1/RSR1 which participates in the regulation of bud-site selection; (4) Human neurofibromitosis (gene NF1). NF1 is associated with type 1 neurofibromatosis, one of the most frequently inherited genetic diseases characterized, in part, by multiple neural tumors. NF1 has been shown genetically and biochemically to interact with and stimulate the GTPase activity of ras; (5) Drosophila Gap1, which acts as a negative regulator of signalling by the Sevenless (SOS) receptor tyrosine kinase involved in eye development. Human SOS1 and SOS2 genes have also been recently identified which encode proteins that control GDP.fwdarw.GTP exchange on ras proteins and are involved in signal transduction by tyrosine kinase receptors. In situ hybridization shows that SOS1 maps to 2p22.fwdarw.p16 and SOS2 to 14q21.fwdarw.q22 in the human genome.
Another important target of ras is raf. The protein encoded by the c-raf-1 protooncogene is thought to function downstream of p21 ras because disruption of raf blocks signalling by ras in a number of systems. A highly-conserved 81 residue region of the N-terminus of raf protein has been shown to be critical as the ras protein interaction region. Importantly, the raf gene product interacts with both wild-type and activated ras protein. In one study, approximately 50% of the clones identified as interacting with ras were encoded portions of the c-raf and A-raf serine/threonine kinases. Thus, ras and the N-terminal region of raf protein associate directly in vitro and this interaction is dependent on GTP bound to ras.
Within the superfamily of ras-related GTP-binding proteins, only the ras protein itself has been shown to act as an oncogenic protein. Many other proteins, however, have substantial amino acid homology to ras. This ras superfamily of GTP-binding proteins (&gt;50 members) regulates a diverse spectrum of intracellular processes. These include cellular proliferation and differentiation, intracellular vesicular trafficking, cytoskeletal control, NADPH oxidase function, as well as others. Some of these homologs may have biological activities which are related to ras. For example, rhoA encodes a ras-related GTP-binding protein that was thought principally to play a role in cytoskeletal organization. Recent evidence, however, has suggested both that rhoA could act either as a dominant oncogene, since transfection of both normal and activated rho genes confer a transformed phenotype on fibroblast cells in culture, or as a recessive tumor suppressor gene, by virtue, in part, of its chromosomal location at 3p21, a site deleted in many human malignancies. Thus, it is important to consider these ras homologs as potentially involved in cell growth and transformation.
Azatyrosine strongly inhibits oncogenic ras-p-21. This small molecule induces the rrg gene, which encodes a proteinase sequence showing 90% amino acid sequence identity to lysyl oxidase.
To acquire transforming potential, the precursor of the ras oncoprotein must undergo farnesylation or similar modification of the cysteine residue located in a carboxyl-terminal tetrapeptide. These C-terminal lipid modifications are essential for the interaction of ras-related proteins with membranes. While all ras proteins are farnesylated and some palmitoylated, the majority of other ras-related proteins are geranylgeranylated. Thus selective peptide and peptidomimetic inhibitors of ras lipidation have found potential utility as anti-oncogenic agents.
In view of the foregoing, there is a longfelt need in the art for agents which inhibit the transforming ability of ras. As described above, selective peptide and peptidomimetic inhibitors or ras lipidation have found potential utility as anti-oncogenic agents (Kohl et al. (1993) Science 260:1934-1937; James et al. (1993) Science 260:1937-1942). Similarly, FR patents 2694296 and 2690162 teach that peptides derived from the GAP protein may serve to inhibit ras. However, neither '694296 nor '690162 describes peptides derived from the ras protein itself. EP 203587 describes new ras oncogene polypeptides which are used for producing antibodies for immunogenic assays. However, these sequences are derived from ras and its homologs in the carboxyl terminal domain (residues 170-189 in SEQ ID NO:5) and are thus physically distant from and completely unrelated to any sequences claimed herein. Furthermore, these sequences were claimed for the production of antibodies, preferably by linking to an immunogenic carrier, and a claim for direct therapeutic application was not made.
Thus, peptides constructed from ras and its homologs for therapeutic application, namely by interfering with downstream or upstream actions of ras itself, are useful. Furthermore, the method of identification of said peptides utilizing calculational approaches is believed novel and has unexpectedly led us to these cyclic peptides and peptidomimetics disclosed herein.